Growth of spoilage bacteria during storage and transport of meat

Transcription

1 SCIENTIFIC OPINION ADOPTED: 8 June 2016 doi: /j.efsa Abstract Growth of spoilage bacteria during storage and transport of meat EFSA Panel on Biological Hazards (BIOHAZ) Pseudomonads and lactic acid bacteria (LAB) are the most relevant organisms for assessing the effect of specific chilling time temperature scenarios on the growth of spoilage bacteria under aerobic and anaerobic (vacuum packs) conditions, respectively. Pseudomonad growth was modelled on beef, pork and lamb carcasses, chilled to specific target surface temperatures and compared with the growth that would be achieved if the carcasses were chilled to a core temperature of 7 C (Regulation (EC) No 853/2004). Pseudomonad growth with the combination of chilling the carcass surface to a target temperature (1 10 C for beef and lamb, and 5 10 C for pork) and transportation at that temperature plus 1 C was also modelled for 1 48 h (assuming an initial count of 1 CFU/cm 2 ). Finally, the growth of pseudomonads and LAB was modelled on meat intended for use in minced meat/meat preparations, stored at temperatures of 1 7 C (inclusive) for 1 12 days. The effect of storage temperature and initial count on the time to reach 10 7 CFU/cm 2 was also investigated. The outputs suggest that chilling bovine or ovine carcasses to between 4 and 10 C surface temperature, inclusive, results in similar or lower predicted pseudomonad growth as compared to chilling to a core temperature of 7 C. The results for porcine carcasses depended on the target surface temperature and chilling curve applied. It was also predicted that pseudomonads and LAB grow steadily on meat stored at 1 7 C and LAB counts exceeded 10 7 CFU/cm 2 when stored for 11 days at 7 C. It was concluded that the time temperature chilling profiles that may be used to obtain similar or less growth to that obtained when chilling to a core temperature of 7 C is dependent on the initial contamination level European Food Safety Authority. EFSA Journal published by John Wiley and Sons Ltd on behalf of European Food Safety Authority. Keywords: carcass chilling, time temperature integration, spoilage, pseudomonads, lactic acid bacteria Requestor: European Commission Question number: EFSA-Q Correspondence: biohaz@efsa.europa.eu EFSA Journal 2016;14(6):4523

2 Panel members: Ana Allende, Declan Bolton, Marianne Chemaly, Robert Davies, Pablo Salvador Fernandez Escamez, Rosina Girones, Lieve Herman, Kostas Koutsoumanis, Roland Lindqvist, Birgit Nørrung, Antonia Ricci, Lucy Robertson, Giuseppe Ru, Moez Sanaa, Marion Simmons, Panagiotis Skandamis, Emma Snary, Niko Speybroeck, Benno Ter Kuile, John Threlfall and Helene Wahlstr om Acknowledgements: The Panel wishes to thank the members of the Working Group on growth of spoilage bacteria during storage and transport of meat: Declan Bolton, Laurent Guillier, and Kostas Koutsoumanis and EFSA staff member: Michaela Hempen for the support provided to this scientific opinion. Suggested citation: EFSA BIOHAZ Panel (EFSA Panel on Biological Hazards), Scientific opinion on the growth of spoilage bacteria during storage and transport of meat. EFSA Journal 2016; 14(6):4523, 38 pp. doi: /j.efsa ISSN: European Food Safety Authority. EFSA Journal published by John Wiley and Sons Ltd on behalf of European Food Safety Authority. This is an open access article under the terms of the Creative Commons Attribution-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited and no modifications or adaptations are made. The EFSA Journal is a publication of the European Food Safety Authority, an agency of the European Union. 2 EFSA Journal 2016;14(6):4523

3 Summary Following a request from the European Commission, the EFSA Panel on Biological Hazards (BIOHAZ) was asked to deliver a scientific opinion on the growth of spoilage bacteria, including Clostridium, on: (1) carcasses using similar time temperature combinations as applied for pathogens in the Scientific Opinion on the public health risks related to the maintenance of the cold chain during storage and transport of meat. Part 1 (meat of domestic ungulates), and to revise these chilling profiles if bacterial growth in excess of that which would be achieved if the carcasses were chilled to a core temperature of 7 C, as required by Regulation (EC) No 853/2004, was predicted and (2) raw meat materials intended for the production of minced meat or meat preparations using the time temperature combinations applied for pathogens in the Scientific Opinion on the public health risks related to the maintenance of the cold chain during storage and transport of meat. Part 2 (minced meat from all species), and to revise these chilling conditions if bacterial growth in excess of that which would be achieved using the current recommendations was predicted. To fulfil this mandate, and based on a review of the scientific literature, pseudomonads were considered to be the main spoilage bacteria under aerobic conditions, i.e. on the carcasses and meat cuts stored in air (aerobically), and lactic acid bacteria (LAB) as the primary spoilage agents on meat cuts stored under anaerobic conditions (vacuum-packed), as is the norm in the red meat industry. Brochothrix thermosphacta may also cause meat spoilage under anaerobic conditions as can psychrophilic Clostridium spp. The latter were specifically requested for consideration in the Terms of Reference. Their growth was not modelled directly as it was considered that LAB, given their higher prevalence and similar growth rate, were a more appropriate target for modelling bacterial growth on meat stored under anaerobic conditions and it was assumed that the predictions for LAB would also apply to Clostridium spp. While it was generally accepted that the vast majority of spoilage bacteria occur on the surface of the carcass, the term within was included to cover Clostridium spp. that cause deep tissue spoilage, such as bone taint. Furthermore, at the specific request of the European Commission, this was expanded to include target carcass surface temperatures in combination with transport at 1 C of this target. The growth of pseudomonads on beef, pork and lamb carcasses under similar time temperature scenarios to those used in the Scientific Opinion on the public health risks related to the maintenance of the cold chain during storage and transport of meat. Part 1 (meat of domestic ungulates) 0 was calculated using available predictive models and compared with the two baseline scenarios used in that opinion; a mean chilling profile and a worst case (slowest decrease in carcass surface temperature) for each animal species, with the exception of lamb for which a mean chilling profile was not available. The growth of both pseudomonads and LAB on meat cuts intended for use in minced meat and meat preparations and stored under aerobic and anaerobic conditions, respectively, was also predicted at alternative scenarios similar to those used in the Scientific Opinion on the public health risks related to the maintenance of the cold chain during storage and transport of meat. Part 2 (minced meat from all species) 0 using available predictive models. Moreover, the time (h) required by pseudomonads and LAB to reach a spoilage level of 10 7 CFU/cm 2 was estimated for various temperature conditions taking initial contamination levels into account. The models used predicted that bovine or ovine carcasses could be chilled to between 1 and 10 C surface temperature, inclusive, without obtaining pseudomonad growth in excess of that, which would be achieved if the carcasses were chilled to a core temperature of 7 C as required by Regulation (EC) No 853/2004. Pseudomonad growth on porcine carcasses was modelled for target surface temperatures of between 5 and 10 C as the chilling curves used did not go below these temperatures. The results for porcine carcasses were different to those for beef and lamb as not all time temperature combinations resulted in lower pseudomonad growth on porcine carcasses as compared with that which would be achieved if the carcasses were chilled to a core temperature of 7 C. Moreover, it is difficult to model the relationship between core and surface temperature of the carcasses. To ensure heat dissipating from the core does not heat the surface, sufficient heat must be removed from the carcass before transportation. For meat cuts intended for minced meat or meat preparations, the growth of pseudomonads on poultry was estimated using the parameters ph 6.5 and water activity (a w ) 0.993, and on red meat, a ph of 5.7 and an a w of The results of this modelling analysis were inconclusive. Whether or not the predicted growth of pseudomonads and LAB (anaerobic conditions) obtained using the time temperature combinations applied for pathogens in the previous opinion (EFSA BIOHAZ Panel, 2014b) was higher or lower than that obtained using current recommendations was dependent on the initial levels of contamination. 3 EFSA Journal 2016;14(6):4523

4 Table of contents Abstract... 1 Summary Introduction Background and Terms of Reference as provided by the European Commission Current requirements EFSA Opinions on public health risks related to the maintenance of the cold chain during storage and transport of meat. Part 1 and Part Additional scientific advice Terms of Reference Additional information Introduction to the assessment Meat spoilage Factors affecting meat spoilage Pseudomonad counts on beef, pork, lamb and poultry carcasses Approach to answering the Terms of Reference Data and methodologies Models for growth of pseudomonads and lactic acid bacteria Assessment Answers to Terms of Reference 1 and Growth of pseudomonads during carcass chilling in the slaughterhouse Results for beef carcasses Results for pork carcasses Results for lamb carcasses Growth of pseudomonads on the carcasses during chilling in the slaughterhouse and transportation CFU/g (h) at various alternative scenarios Answers to Terms of Reference 3 and Conclusions References Abbreviations Appendix A Baseline scenarios for chilling of beef, lamb and pork EFSA Journal 2016;14(6):4523

5 1. Introduction 1.1. Background and Terms of Reference as provided by the European Commission Current requirements The maintenance of the cold chain is one of the main principles and basic requirements of European Union (EU) legislation on food hygiene. 1 Raw materials, ingredients, intermediate products and finished products that are likely to support the growth of pathogenic microorganisms and/or spoilage bacteria, are to be kept at temperatures that do not result in a risk to health. The cold chain must not be interrupted. In the case of meat (including fresh meat, meat products, minced meat and meat preparations), EU legislation (Regulation (EC) No 853/2004) lays down specific requirements for its storage and transport regarding temperatures and maximum times of storage. Such requirements include: Fresh meat from animals other than poultry: Post-mortem inspection must be followed immediately by chilling in the slaughterhouse to ensure a temperature throughout the meat of not more than 3 C for offal and 7 C for other meat, along a chilling curve that ensures a continuous decrease in the temperature. However, meat may be cut and boned during chilling in establishments attached to the slaughterhouse. Apart from few exceptions, meat must reach these temperatures specified above before transport, and remain at that temperature during transport. The maximum storage time between slaughter and production of minced meat and meat preparations is no more than 6 days and no more than 15 days from the slaughter of the animals in the case of boned, vacuum-packed beef and veal. Poultry meat: After post-mortem inspection, slaughtered animals must be chilled to not more than 4 C as soon as possible, unless the meat is cut while warm in establishments attached to the slaughterhouse. The maximum storage time between slaughter and production of minced meat and meat preparations is no more than 3 days. Minced meat, meat preparations: Immediately after production, minced meat and meat preparations must be wrapped or packaged and be chilled to an internal temperature of no more than 2 C for minced meat and 4 C for meat preparations, or frozen to an internal temperature of not more than 18 C. Minced meat and meat preparations must comply with the microbiological criteria laid down in Regulation (EC) No 2073/ as regards Salmonella, aerobic colony counts and Escherichia coli. These criteria remain applicable EFSA Opinions on public health risks related to the maintenance of the cold chain during storage and transport of meat. Part 1 and Part 2 The European Food Safety Authority (EFSA) adopted the Scientific Opinion on the public health risks related to the maintenance of the cold chain during storage and transport of meat, Part 1 and Part 2, in 2014 (EFSA BIOHAZ Panel, 2014a,b). Part 1 of the Opinion (EFSA BIOHAZ Panel, 2014a) concluded that it is possible to commence carcass transport before a target temperature is reached, without significantly increasing the risk linked to the growth of potentially harmful microorganisms, as long as the carcass temperature continues to decrease towards the target one during transportation. It also concluded that it is possible to develop different combinations of carcass surface target temperature at loading time, with transport time air temperature combinations that ensure pathogen growth is no greater than that 1 Article 4(3)(d) of Regulation (EC) No 852/2004 of the European Parliament and of the Council of 29 April 2004 on the hygiene of foodstuffs. OJ L 139, , p Commission Regulation (EC) No 2073/2005 of 15 November 2005 on microbiological criteria for foodstuffs. OJ L 338, , p EFSA Journal 2016;14(6):4523

6 achieved using the current chilling requirements. The opinion did not address the possible growth of spoilage bacteria. Part 2 of the Opinion (EFSA BIOHAZ Panel, 2014b) recommended a number of time temperature combinations for the storage of fresh meat prior to mincing, all of which gave the same or less bacterial growth than is possible under current legislative requirements. The opinion did not address the possible growth of spoilage bacteria Additional scientific advice After discussions with Member States experts and stakeholders, the Commission considered that further scientific advice is needed as regards the growth of spoilage bacteria in meat (carcases, cuts) during transport after slaughter, and in raw materials for minced meat and meat preparations. The purpose of the request is to ensure compliance with Article 14(5) of Regulation (EC) No 178/ laying down that, in determining whether any food is unfit for human consumption, regard shall be had to whether the food is unacceptable for human consumption according to its intended use, for reasons of contamination, whether by extraneous matter or otherwise, or through putrefaction, deterioration or decay. The current requirement to chill meat immediately after post-mortem inspection remains applicable. In addition, the core temperature currently applicable will have to be reached as soon as possible after the transport stage. Moreover, a need for a transport regime extending for up to 2 or 3 h for minimally chilled meat has been identified. Stakeholders have indicated that a time temperature combination resulting in a transport regime of 1 h (combination e in the recommendations of Part I of the Opinion) is somewhat short for practical purposes, as loading of the lorry can take up to 2 h. Therefore, it would be helpful if other options could be explored that might result in a combination of time and temperature which would result in transport of 2 or 3 h with minimal chilling. This could facilitate the short-term transport of minimally chilled meat to cutting/boning plants relatively close to the slaughterhouse Terms of Reference EFSA is asked to provide a Scientific Opinion on the growth of spoilage bacteria, including Clostridium, in meat and in raw materials stored for minced meat and meat preparations as a consequence of applying flexibility in the maintenance of the cold chain during storage and transport of meat. In particular, EFSA is requested: In relation to transport of meat of domestic ungulates: 1) to investigate the growth of spoilage bacteria both within and on the surface of meat carcasses or parts thereof during storage and transport using the same combinations of temperature and time conditions applied for pathogens adopted in the Scientific Opinion on the public health risks related to the maintenance of the cold chain during storage and transport of meat. Part I ; 2) to revise, if needed, based on the outcome of Term of Reference (ToR) 1, the recommendations for maximum surface temperature and maximum transport time that would give equivalent growth to current requirements to ensure that the recommendations address at the same time both pathogens (as concluded in the Scientific Opinion on the public health risks related to the maintenance of the cold chain during storage and transport of meat. Part 1 0 ) and spoilage bacteria. Recommendations on short distance transport (2 3 h) should be included. In relation to the production of minced meat from all species: 3) to investigate the growth of spoilage bacteria during storage of meat raw materials intended for the production of minced meat or meat preparations using the same combinations of temperature and time conditions for pathogens adopted in the Scientific Opinion on the public health risks related to the maintenance of the cold chain during storage and transport of meat. Part 2 ; 3 Article 14(5) of Regulation (EC) No 178/2002 of the European Parliament and of the Council of 28 January 2002 laying down the general principles and requirements of food law, establishing the European Food Safety Authority and laying down procedures in matters of food safety. OJ L 31, , p EFSA Journal 2016;14(6):4523

7 4) to revise, if needed, based on the outcome of the ToR 3, the recommendations for maximum storage time that would give equivalent growth to current requirements to ensure that the recommendations address at the same time both pathogens (as concluded in the Scientific Opinion on the public health risks related to the maintenance of the cold chain during storage and transport of meat. Part 2 0 ) and spoilage bacteria. In addition, at a later stage, the European Commission requested additional aspects to be addressed in this opinion, specifically: 5) to model the growth of spoilage bacteria at lower temperature transport regimes; 6) to investigate the growth of spoilage bacteria at higher temperature (7, 8, 9 and 10 C) transport regimes Additional information Introduction to the assessment In June 2014, EFSA published the Scientific Opinion on the public health risks related to the maintenance of the cold chain during storage and transport of meat. Part 1 (meat of domestic ungulates) (EFSA BIOHAZ Panel, 2014a). This opinion concluded that (1) Salmonella spp., verocytotoxigenic E. coli (VTEC), Listeria monocytogenes and Yersinia enterocolitica are the most relevant microbial pathogens when assessing the effects of beef, pork and lamb carcass chilling regimes on the potential risk to public health, and (2) most bacterial contamination occurs on the surface of the carcass. Moreover, it provided combinations of maximum surface temperatures at carcass loading and maximum chilling and transport times, resulting in pathogen growth equivalent to or less than that obtained when carcasses are chilled to a core temperature of 7 C in the slaughterhouse. Part 2 of this opinion (EFSA BIOHAZ Panel, 2014b) covered minced meat and investigated the impact of storage time between slaughter and mincing on bacterial pathogen growth using predictive modelling. It concluded that time temperature combinations, other than that legally required by Regulation (EC) No 853/2004 (red meat carcasses must be immediately chilled after post-mortem inspection to not more than 7 C throughout, and that this temperature be maintained until mincing, which must take place not more than 6 or 15 (vacuum-packed meat) days after slaughter), for the storage of fresh meat between slaughter and mincing are possible without increasing bacterial pathogen growth. Moreover, maximum times for the storage of fresh meat intended for minced meat preparation were provided for different storage temperatures. Neither Part 1 nor Part 2 of the previous opinions (EFSA BIOHAZ Panel, 2014a,b) considered the impact of bacterial spoilage on the alternative storage time temperature combinations. This follow-up opinion investigates the growth of spoilage bacteria on the surface of the carcass (aerobic conditions) during chilling and subsequent transportation, using an extended range of time temperature combinations, including those applied to pathogens in the Scientific Opinion on the public health risks related to the maintenance of the cold chain during storage and transport of meat. Part 1 (meat of domestic ungulates) (EFSA BIOHAZ Panel, 2014a) and provides recommendations for the maximum surface temperature and maximum transport time that would give equivalent growth to current requirements (Regulation (EC) No 853/2004). This opinion also investigates the growth of spoilage bacteria on raw meat materials used for minced meat (red meat and poultry) preparation during chilling and subsequent transportation using the same time temperature combinations applied to pathogens in the opinion mentioned above (EFSA BIOHAZ Panel, 2014b), with a view to providing recommendations for maximum storage time, that would give equivalent growth to that which would be achieved based on the requirements in Regulation (EC) No 853/ Meat spoilage Beef, pork and lamb carcasses are chilled immediately after post-mortem inspection in slaughterhouse chilling rooms (Figure 1). As the carcasses are exposed to air this process is aerobic. After h of chilling, the carcasses are typically moved to a boning hall where they are cut into primary cuts called primals. These are typically stored for up to 6 weeks in vacuum packs under 7 EFSA Journal 2016;14(6):4523

8 anaerobic conditions. Ground meat products may be prepared from trimmings from deboning or trimmings from primals after 6 weeks anaerobic storage. These may then be stored aerobically or anaerobically. Figure 1: The chilling and chilled storage conditions used for beef, pork and lamb carcasses and associated primals and trimmings Chilling red meat and poultry carcasses is essential to retard bacterial growth. Chilling is also required for appearance and eating quality. Most carcasses are refrigerated using a system based on forced convection air chilling (James and James, 2004), although spray chilling may also be used. Spray chilling is faster than air chilling and operates on the same principle as air chilling except potable water (instead of air) is chilled before being applied to the carcasses as a fine spray. It is primarily used in poultry, but may also be used in beef, pork and lamb processing plants (Brown and James, 1992; Brown et al., 1993; James and James, 2004). A more thorough review of meat chilling methods is provided in the Scientific Opinion on the public health risks related to the maintenance of the cold chain during storage and transport of meat. Part 1 (meat of domestic ungulates) (EFSA BIOHAZ Panel, 2014a). Regulation (EC) No 853/2004 requires that carcasses are immediately chilled after post-mortem inspection to ensure that the temperature throughout the meat is not higher than 7 C in the case of meat, and not higher than 3 C for offal. However, there is no provision on the time limit by when this temperature must be achieved. Moreover, beef and lamb carcasses are usually not chilled to below 10 C (core temperature) within the first 10 h to avoid cold shortening and toughening of the meat. Thus, bacteria will grow on the surface of the carcass until the temperature is reduced sufficiently to retard bacterial activity. Meat is considered to be spoiled when discolouration, off-odour and/or slime develop and is usually caused by bacteria (Tsigarida and Nychas, 2001). Pseudomonads, Lactobacillus and Enterococcus, for example, produce slime on meat. Enterococcus may also produce hydrogen peroxide greening similar to hydrogen sulfide greening caused by Clostridium spp. The growth of bacteria on meat is influenced by temperature, ph, water activity, nutrient availability, storage atmosphere and competition from other organisms and small changes in these factors can greatly influence spoilage (Sumner and Jenson, 2011). Although indigenous enzymes may also be involved, their contribution is considered to be negligible compared with bacterial action (Tsigarida and Nychas, 2001). The bacteria commonly found on fresh meat belong to a range of different genera, including Achromobacter, Acinetobacter, Aeromonas, Alcaligenes, Alteromonas, Arthrobacter, Bacillus, Campylobacter, Carnobacterium, Citrobacter, Clostridium, Corynebacterium, Enterobacter, Escherichia, Flavobacterium, Hafnia, Klebsiella, Kluyvera, Kocuria, Kurthia, Lactobacillus, Lactococcus, Leuconostoc, Listeria, Microbacterium, Micrococcus, Moraxella, Paenibacillus, Pantoea, Proteus, Providencia, Pseudomonas, Shewanella, Staphylococcus, Streptococcus, Vibrio, Weissella and Yersinia (Nychas et al., 2007). These organisms originate from the animal (hide, fleece or skin and intestines) and the abattoir environment. The main spoilage defects and causal bacteria are shown in Table EFSA Journal 2016;14(6):4523

9 Table 1: The main spoilage defects and causal bacteria (adapted from Nychas et al., 2008) Defect Meat product Causal bacteria Slime Fresh meat Pseudomonads, Lactobacillus, Enterococcus, Weissella and Brochothrix Hydrogen peroxide Fresh meat Weissella, Leuconostoc, Enterococcus and Lactobacillus greening Hydrogen sulfide Vacuum-packed fresh meat Shewanella and Clostridium greening Hydrogen sulfide Cured meats Vibrio and Enterobacteriaceae production Sulfide odour Vacuum-packed fresh meat Clostridium and Hafnia Cabbage odour Bacon Providencia Cheesy or dairy odour Vacuum-packed fresh meat Brochothrix thermosphacta Putrefaction Ham Enterobacteriaceae and Proteus Bone taint Whole meats Clostridium and Enterococcus Souring Vacuum-packed meats Lactic acid bacteria, Enterococcus, Micrococcus, Bacillus and Clostridium The dominant spoilage genera (and species within a given genus) are determined by the storage conditions. Chilled storage selects for psychrophilic and psychrotrophic bacteria. Under aerobic conditions, the spoilage consortium of bacteria is usually dominated by pseudomonads (Stanbridge and Davis, 1998; Koutsoumanis et al., 2006). Three species of Pseudomonas, Pseudomonas fragi, Pseudomonas fluorescens and Pseudomonas lundensis, are primarily responsible for the formation of slime and off-odour, usually when their population reaches colony forming units (CFU)/cm 2 (Nychas et al., 2008). Enterobacteriaceae, especially cold-tolerant species, such as Hafnia alvei, Serratia liquefaciens and Pantoea agglomerans, are also commonly found on fresh meat but rarely contribute to spoilage unless there is temperature abuse (Nychas et al., 1998). Lactic acid bacteria (LAB) and Brochothrix thermosphacta are oxygen-tolerant anaerobes and are commonly detected on aerobically stored chilled meat, but are not considered as major spoilage contributors, with the possible exception of lamb (Holzapfel, 1998). These bacteria are, however, usually the predominant spoilage organisms of meat stored under anaerobic conditions (e.g. vacuum-packed or modified atmosphere). This type of spoilage, which typically occurs when maximum numbers (10 8 CFU/cm 2 ) are achieved (Jones, 2004), is characterised by souring rather than putrefaction. Chilled meat stored under anaerobic conditions may also be spoiled by a range of psychrotolerant/ psychrophilic Clostridium spp. These Clostridium spp. grow relatively slowly and spoilage typically occurs in correctly chilled batches (0 2 C) after 4 6 weeks, characterised by a putrid smell (H 2 S) with a metallic sheen on the meat, with or without gas production. Clostridium algidicarnis, Clostridium frigoris, Clostridium bowmanii, Clostridium frigidicarmis and Clostridium ruminantium have been associated with spoilage without gas production (Broda et al., 1999, 2000; Adam et al., 2010; Cavill et al., 2011), while other species, such as Clostridium estertheticum and Clostridium gasigenes, produce large volumes of gas, primarily carbon dioxide (Moschonas et al., 2010; Yang et al., 2011). The packs inflate and eventually burst; thus, this type of spoilage is often referred to as blown pack spoilage (Bolton et al., 2015). Clostridium spp. are also the primary causative agents of deep tissue spoilage of meat, including bone taint Factors affecting meat spoilage Factors affecting meat spoilage include temperature, ph, water activity and storage atmosphere. During chilling, the temperature of the surface of the carcass changes (EFSA BIOHAZ Panel, 2014a). Meat is usually stored at temperatures of around 2 C (James and James, 2004), with the exception of meat being transported long distances where a temperature of 1.5 C is recommended (Jeremiah and Gibson, 2001). Small changes in temperature can significantly affect the shelf-life. Thus, increasing the temperature from 1.5 to 0, 2 or 5 C, will decrease the time to spoilage by approximately 30%, 50% and 70%, respectively (Sumner and Jenson, 2011). The ph of the muscle is approximately 7.0 at slaughter and decreases to ph from 5.3 to 5.8 over h in beef, and 6 12 h in pork. In lamb carcasses, this usually occurs in approximately 24 h 9 EFSA Journal 2016;14(6):4523

10 (McGeehin et al., 1999). Dark firm dry (DFD) meat can occur in all species but is more common in beef. DFD is the result of pre-slaughter stress and the depletion of glycogen reserves to below 0.6%, and DFD meat has a ph of This higher ph promotes the growth of spoilage bacteria. Fresh meat has a water activity (a w ) of approximately 0.99 (ICMSF, 1998), which decreases during chilling to approximately (Reid et al., 2015). Thus, a wide range of bacteria are able to survive and grow on meat and carcass surfaces Pseudomonad counts on beef, pork, lamb and poultry carcasses The levels of bacterial contamination, including pseudomonads on beef, pork, lamb and poultry carcasses will depend on a variety of factors including season, animal/bird cleanliness, abattoir prerequisite activities especially good hygiene practices (GHP), sampling stage, etc. Thus, pseudomonad carcass counts may vary considerably not only within a given abattoir over time but also between abattoirs. The currently available data are presented in Table 2. Relatively few studies have reported pseudomonad counts on beef carcasses. A recent Irish study investigated pseudomonad levels on beef carcasses immediately pre-chill and in the first 96 h in the chillers. In this study, 10 carcasses were tested as per Commission Decision 2001/471/EC 4 ; the neck, brisket, flank and rump (100 cm 2 of each) were sampled on each carcass using a sterile cellulose acetate sponge. The experiment was repeated on three separate occasions in the same commercial beef export abattoir. The mean pseudomonad count immediately before chilling was 1.14 log 10 CFU/cm 2. Over the first 48 h, this count decreased by 0.11 log 10 CFU/cm 2 and then increased by 0.83 log 10 CFU/cm 2 to a final count of 1.86 log 10 CFU/cm 2 between 48 and 96 h (Reid et al., 2015). Lasta et al. (1995) reported an initial pseudomonad count of 3.2 log 10 CFU/cm 2 on beef briskets, which increased to 8.9 log 10 CFU/cm 2 after 14 days storage at 5 C. The pseudomonad count on Romanian beef carcasses ranged from 1.04 to 5.48 log 10 CFU/cm 2 in a study of 18 carcasses (Dan et al., 2003). In contrast, Gustavsson and Borch (1993) found very low counts (0.4 log 10 CFU/cm 2 ) on the brisket, lateral forerib and foreleg during chilling. Table 2: Pseudomonad counts on beef, pork and lamb carcasses reported in the scientific literature Carcass type Beef Data provided Mean count of 1.14 (range ) log 10 CFU/cm 2 (4 sites sampled as per Commission Decision 2001/471/EC) immediately before chilling increasing to 1.86 log 10 CFU/cm 2 after 96 h chilling Reference Reid et al. (2015) 3.2 log 10 CFU/cm 2 (briskets) immediately before chilling increasing to Lasta et al. (1995) 8.9 log 10 CFU/cm 2 after 14 days storage at 5 C log 10 CFU/cm 2 Dan et al. (2003) 0.4 log 10 CFU/cm 2 Gustavsson and Borch (1993) Pork log 10 CFU/cm 2 Dan et al. (2005) 4.37 (neck/chest), 4.49 (thigh), 5.45 (lateral abdominal) and 4.55 Sala et al. (2010) (coccygeal region) log 10 CFU/cm 2 Lamb 3.11 (after fleece removal), 3.09 (after evisceration) and 3.08 (after Bhandare et al. (2007) washing) log 10 CFU/cm log 10 CFU/cm 2 (leg/flank regions) Sauter et al. (1980) Poultry log 10 CFU/cm 2, post-chill Vareltzis et al. (1997) 1.8 (before scalding), 1.7 (after scalding) and 3.1 after de-feathering) Geornaras et al. (1997) log 10 CFU/g of neck skin Mean count of 3.96 log 10 CFU/cm 2 (ranging from 0.44 to log 10 CFU/cm 2 Holder et al. (1997) Pseudomonad counts have also been reported for pork, and to a lesser extent lamb carcasses. In two different Romanian studies, the pseudomonad count on pork carcasses ranged from 2.74 to 4 Commission Decision 2001/471/EC of 8 June 2001 laying down rules for the regular checks on the general hygiene carried out by the operators in establishments according to Directive 64/433/EEC on health conditions for the production and marketing of fresh meat and Directive 71/118/EEC on health problems affecting the production and placing on the market of fresh poultry meat. OJ L 165, , p EFSA Journal 2016;14(6):4523

11 6.57 log 10 CFU/cm 2 (Dan et al., 2005), while Sala et al. (2010) obtained counts of 4.37, 4.49, 5.45 and 4.55 log 10 CFU/cm 2 on the neck/chest, thigh, lateral abdominal region and the coccygeal region of pork carcasses. Bhandare et al. (2007) reported mean Pseudomonas aeruginosa counts on Indian sheep/goat carcasses of 3.11 log 10 CFU/cm 2 after flaying (hide/fleece removal), 3.09 log 10 CFU/cm 2 after evisceration and 3.08 log 10 CFU/cm 2 after washing in the abattoir. Interestingly, Sauter et al. (1980) had previously obtained similar counts, 3.32 and 3.51 log 10 CFU/cm 2, respectively, on the leg and flank regions of lamb carcasses in the USA. There is also very limited data available for poultry carcasses. Vareltzis et al. (1997) obtained pseudomonad counts of log 10 CFU/cm 2 on poultry carcasses post-chilling. Geornaras et al. (1997) reported pseudomonad counts of 1.8, 1.7 and 3.1 log 10 CFU/g on neck skin before scalding, after scalding and after defeathering, respectively. Holder et al. (1997) obtained a mean pseudomonad count on whole carcasses of 3.96 log 10 CFU/cm 2. These ranged from 0.44 log 10 CFU/cm 2 on the medial breast to 4.45 log 10 CFU/cm 2 on the lateral drumstick during portioning of the carcass Approach to answering the Terms of Reference The objective of the ToRs was to assess the impact of the time temperature chilling profiles proposed as alternatives to the current legislation (Regulation (EC) No 853/2004) in the Scientific Opinion on public health risks related to the maintenance of the cold chain during storage and transport of meat. Part 1 on the growth of spoilage bacteria. Pseudomonads were selected for modelling bacterial spoilage of beef, pork and lamb carcasses stored aerobically because they are considered to be the main spoilage organisms under aerobic chilling conditions, and also because there were suitable models available. LAB were selected as the target organism for modelling meat stored under anaerobic conditions (e.g. vacuum-packed meat primals) as under anaerobic chilled conditions they are assumed to be the major spoilage organism, and again because suitable models were available to predict growth. Although psychrophilic Clostridium spp. and Br. thermosphacta may also cause spoilage of anaerobically stored meat cuts and the former were mentioned in the ToRs, these were not included in the analysis as it was assumed that their growth would be similar or slower than that of LAB (Mejlholm and Dalgaard, 2013). The growth of pseudomonads at various alternative time temperature scenarios during chilling and transportation was calculated, based on available predictive models, and compared with the two baseline scenarios (mean chilling profile and worst-case chilling profile, as per the Scientific Opinion on the public health risks related to the maintenance of the cold chain during storage and transport of meat. Part 1 (meat of domestic ungulates) ) for each animal species. For comparison, growth was expressed as log 10 CFU/cm 2. The time (h) required by pseudomonads and LAB to reach a spoilage level of 10 7 CFU/cm 2 was estimated for the same combinations of temperature and time conditions applied for pathogens adopted in the aforementioned opinion (EFSA BIOHAZ Panel, 2014a). 2. Data and methodologies The general approach proposed is based on the change in the temperature kinetics in terms of the bacterial growth potential using predictive microbiology models. The approach is also called time temperature integration or temperature function integration (McMeekin, 2007). This approach is widely used and has been applied in previous EFSA opinions (EFSA BIOHAZ Panel, 2014a,b). Other examples illustrating the use of time temperature integration in the area of meat refrigeration include Gill and Jones (1997), Dickson et al. (1993), Jericho et al. (1998) and Lovatt et al. (2006), all of which have used records of the temperatures at the surface and/or core of beef, pork and/or lamb carcasses. These temperature records, coupled with growth models made it possible to estimate microbial growth during chilling. The associated studies facilitate validation of the consequences of specific refrigeration methods (e.g. spraying during cooling, effect of passing through a refrigeration tunnel, etc.). Time temperature integration has previously been applied in meat industry hazard analysis and critical control point (HACCP) plans. In Australia, for example, on the basis of a model predicting the rate of growth of E. coli as a function of the temperature, water activity and the lactate concentration (Mellefont et al., 2003), the Australian Quarantine Inspection Service (AQIS) has established the rules for the chilling of carcasses for export. 5 Using recorders placed in the carcasses during chilling, the growth of E. coli can be calculated and expressed in the form of a refrigeration index. The values 5 Available online: EFSA Journal 2016;14(6):4523

12 calculated for each slaughter site must comply with values that may not be exceeded, to verify that the required refrigeration was applied to the carcasses. More specifically, the output of time temperature integration can be used in two different ways. In the first approach, the initial level of contamination is not considered, i.e. growth potential rather than final levels are calculated. The growth potential associated with a time temperature is then compared with a growth potential obtained in a reference situation (e.g. EFSA BIOHAZ Panel, 2014a,b) or to a target growth potential (e.g. less than a doubling, AFSCA, 2008). In the second approach, the initial level of the bacteria of interest is taken into account. Time temperature integration is then used to calculate the time to reach a target level given an initial level. ANSES (2010) used this second approach for defining the maximum delay time for entry into the cold rooms related to failure of the slaughter chain. Both the initial level and growth of pseudomonads were considered. The maximum times proposed were directly linked to the initial level of contamination that is specific to the general hygiene of each slaughterhouse; the lower the initial contamination, the higher the delay time. This approach is thus particularly appropriate for bacteria for which a high variability of initial contamination occurs. As contamination levels on carcasses or meat cuts are highly variable for pseudomonads or LAB (see e.g. Augustin and Minvielle, 2008; Ghafir et al., 2008), both approaches were used. Pseudomonas and LAB are considered the specific spoilage organisms (SSO) of meat during storage under aerobic conditions and in vacuum packs, respectively. The growth of pseudomonads at various alternative scenarios during chilling and transportation was calculated based on available predictive models (Figure 2). To address ToRs 1 and 2, the alternative scenarios are compared with two baseline scenarios (mean and worst case, described below) for beef and pork and the worst-case scenario for lamb. Predicted growth is defined as the difference between the final concentration and a starting point equal to 1 CFU/cm 2. The time (h) required by pseudomonads to reach a spoilage level of 10 7 CFU/cm 2 was estimated for the same combinations of temperature and time conditions applied for pathogens adopted in the Scientific Opinion on the public health risks related to the maintenance of the cold chain during storage and transport of meat. Part 1 (meat of domestic ungulates) (EFSA BIOHAZ Panel, 2014a). Figure 2: Pseudomonad growth was estimated for three different combinations of surface and time (h) In order to model the growth of pseudomonads, the same surface temperature time baseline scenarios representing the current situation as those in the previous opinion (EFSA BIOHAZ Panel, 2014a) were applied. Using beef and pork carcass surface temperature data obtained in commercial slaughterhouse chillers, two chilling profiles were developed; a mean profile calculated from the mean surface temperatures at various time points during carcass chilling and a worst-case profile calculated using the highest temperatures recorded at each time point. As there was insufficient data available on the changes in surface temperature of lamb carcasses during chilling, it was not possible to obtain a mean profile and, based on expert opinion, the chilling profile obtained was considered to be a worst-case profile. The commercial chilling data also included data on the core temperature during carcass chilling, thus the time to reach 7 C, which marks the end of the chilling process, was obtained EFSA Journal 2016;14(6):4523

13 When modelling pseudomonad growth on carcasses during chilling in the slaughterhouse, the mean and worst-case temperature profiles were used for beef and pork, and the worst-case profile for lamb. In order to model pseudomonad growth during the combination of chilling in the slaughterhouse and chilled storage during transport, two scenarios were developed; 1) The mean baseline scenario (beef and pork) was the calculated mean surface temperature profile during chilling in the slaughterhouse when the carcasses were chilled to a core temperature of 7 C and transportation at a constant surface temperature of 4 C for 48 h. 2) The worst-case baseline scenario (beef, pork and lamb) was the surface temperature profile composed of the highest temperature obtained at each time point during chilling in the slaughterhouse when the carcasses were chilled to a core temperature of 7 C and transportation at a constant surface temperature of 4 C for 48 h (Appendix A). To address ToRs 3 and 4, the time (h) required by pseudomonads and LAB to reach a spoilage level of 10 7 CFU/cm 2 during storage was estimated for various temperature conditions and initial pseudomonads or LAB levels Models for growth of pseudomonads and lactic acid bacteria Several secondary models for predicting growth rate of pseudomonads are available (e.g. Gill and Jones, 1992; Neumeyer et al., 1997; Pin and Baranyi, 1998; Dominguez and Schaffner, 2007). These models were established using growth rate data obtained in broth medium and/or meat. All of these models consider the effect of temperature. Growth of pseudomonads during carcass chilling and transportation was estimated using the ComBase model (available at This model was selected because differences of predicted growth rates between models are limited (Baranyi et al., 1999; ANSES, 2010) and because the ComBase model includes the effect of ph and water activity. Several secondary models for predicting growth rate of LAB are available (e.g. Devlieghere et al., 2000; Wijtzes et al., 2001; Mejlholm and Dalgaard, 2013). Growth of LAB was estimated using the Food Spoilage and Safety Predictor (FSSP TM ) v. 4.0 growth model (available at The FSSP TM is based on the Mejlholm and Dalgaard (2013) model for psychrotrophic LAB. The selection of this model was based on the fact that the most extensive validation has been carried out for this model. The models and the assumed environmental conditions used in the growth predictions for pseudomonads and LAB are shown in Tables 3 and 4. The assumption of the lag phase absence, together with the assumed high a w and ph, as well as no competition from other meat bacterial flora, represents conditions that are favourable for the growth of pseudomonads and LAB and most likely results in an overestimation of growth. However, as the approach used is based on the comparison of temperature scenarios, this is not expected to affect the results and conclusions. Table 3: Characteristics of ComBase (a) growth model for pseudomonads Model Pseudomonads Primary growth model Baranyi and Roberts (1994) Secondary growth model Polynomial Environmental parameters in model Temperature, salt, a w Product validation studies Unknown Range of applicability Temperature (0 20 C), a w ( ), ph ( ) a w : water activity. (a): Table 4: Characteristics of FSSP TM v. 4.0 (a) growth model for LAB Model LAB growth and growth boundary models Primary growth model Logistic model with delay Secondary growth Simplified cardinal parameter type model model Environmental parameters in model Temperature, atmosphere (CO 2 ), water phase salt/a w, ph, smoke components/phenol, nitrite and organic acids in water phase of product (acetic acids, benzoic acid, citric acid, diacetate, lactic acid and sorbic acid) 13 EFSA Journal 2016;14(6):4523

14 Model Product validation studies Range of applicability LAB growth and growth boundary models The model has been extensively validated using data from fresh and processed seafood and meat products (Mejlholm and Dalgaard, 2007, 2013, 2015) Temperature (0 25 C), atmosphere (0 100% CO 2 ), water phase salt ( %), ph ( ), smoke components/phenol ( ppm), nitrite (0 209 ppm in product), acetic acid (0 12,600 ppm in water phase), benzoic acid (0 1,800 ppm in water phase), citric acid (0 7,300 ppm in water phase), diacetate (0 3,000 ppm in water phase), lactic acid (0 67,000 ppm in water phase) and sorbic acid (0 1,600 ppm in water phase) FSSP: Food Spoilage and Safety Predictor; LAB: lactic acid bacteria; a w : water activity. (a): The above models were used to predict growth of pseudomonads and LAB assuming a worst-case scenario based on growth without lag phase and optimum conditions for growth for meat characteristics, i.e. ph = 6.5, a w = or water phase salt (WPS) = 1. The shelf-life of meat (time to spoilage) was estimated as the time required by pseudomonads or LAB to reach a spoilage level of 10 7 CFU/g. For ToRs 3 and 4, the growth of pseudomonads on poultry was estimated using the parameters ph 6.5 and a w 0.993, and on red meat, a ph of 5.7 and an a w of The outputs of these modelling exercises are presented as summarised in Table 5. Tables 6 10 present the predicted growth of pseudomonads on beef, pork and lamb carcasses during chilling in the abattoir immediately after slaughter and dressing. Tables present the predicted growth of pseudomonads on beef, pork and lamb carcasses during the combination of chilling in the abattoir and chilled transportation. Tables model the growth of pseudomonads or LAB on red meat and/or poultry stored at a range of temperatures for up to 12 days. The format for the tables predicting the effect of the combination of time during chilling (to a specific target carcass surface temperature) and transport on bacterial count has been changed to that used in the opinion covering the growth of pathogenic bacteria. Instead of predicting the time required to reach a given bacterial concentration equivalent to that which would be achieved if the carcass was chilled to a core temperature of 7 C and transported at that temperature, the format has been changed to predict the time required to reach 10 7 pseudomonads/cm 2 (end of shelf-life). This was done to directly address the ToRs in each opinion. Thus, the revised format, with shelf-life as the key metric, was considered to be more appropriate for addressing the ToRs in this opinion. Table 5: An overview of the predictive modelling performed Table Assumed initial count Carcass/ meat Chilling profile Target surface temperature during chilling Temperature During transportation Objective Predicted growth of pseudomonads on carcasses during chilling in the abattoir 6 1 CFU/cm 2 Beef M 1 10 C NA To compare the 7 1 CFU/cm 2 Beef WC 1 10 C NA 8 1 CFU/cm 2 Pork M 1 10 C NA 9 1 CFU/cm 2 Pork WC 1 10 C NA 10 1 CFU/cm 2 Lamb WC 1 10 C NA Predicted growth of pseudomonads during transportation in a refrigerated vehicle 11 1 CFU/cm 2 Beef, pork and lamb predicted growth with that which would be achieved if the carcass was chilled to a core of 7 C (as per Reg. (EC) No 853/2004) NA NA 5 10 C To predict the growth of pseudomonads during transportation log 10 CFU/cm 2 Beef M & WC 1 C 0 2 C To predict the time (h) log 10 CFU/cm 2 Beef M & WC 2 C 1 3 C until the pseudomonad log count reaches 10 CFU/cm 2 Beef M & WC 3 C 2 4 C 10 7 CFU/cm 2 (end of log 10 CFU/cm 2 Beef M & WC 4 C 3 5 C shelf-life) log 10 CFU/cm 2 Beef M & WC 5 C 4 6 C log 10 CFU/cm 2 Beef M & WC 6 C 5 7 C 14 EFSA Journal 2016;14(6):4523